Pyrosequencing #sequencing #dna

Pyrosequencing enables real-time DNA sequencing based on the detection of pyrophosphate released during DNA elongation. A specific pair of PCR primers, one of them biotin labeled, is used to generate a locus-specific amplicon of approximately 200 base pairs (bp) while a sequencing primer is used to sequence the region of interest and quantify heteroplasmy levels. The procedure requires denaturation of the double-stranded PCR products and isolation of biotin-linked single strands with streptavidin-coated beads to be used as template for pyrosequencing. After annealing of the sequencing primer, nucleotides are added one at a time into the reaction mix. If the complementary nucleotide is incorporated into the nascent elongating strand, pyrophosphate is released and converted to ATP by ATP sulfurylase. This ATP along with oxygen is used by the luciferase to convert luciferin into oxyluciferin in a reaction that generates light and releases pyrophosphate Light production is proportional to the amount of incorporated nucleotide, and it is represented as a pyrogram peak that provides information that can be used to quantify heteroplasmy levels. please watch video for more detail